Most neuropathies, e.g. nerve injury caused by a traffic accident, nerve injury or nerve degeneration caused by remedies for cancers or AIDS, and the injury or hypofunction of peripheral nerves or central nerves caused by amyotrophic lateral sclerosis, diabetic neuropathy, dementia senilis, Alzheimer's disease, Parkinson's disease and the like are intractable, they present a serious condition, and often lead to death of a patient. However, at present, there is no effective remedy. Since, degeneration and deciduation of nerve tissues, transaction and regression of axons, and so on occur in these neuropathies, in order to prevent and treat the neuropathy, a factor which acts to inhibit nerve tissue degeneration or apoptosis and promoting axons regeneration, is required as an effective remedy.
Since Levi-Montalcini et al. found nerve growth factor (NGF) about 40 years ago, it has been revealed that humoral factors acting on nerve cells, i.e., a neurotrophic factor group including ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and glia-derived neurotrophic factor (GDNF), and cytokines or the like contain a factor acting to support the survival of various neurons and to regenerate axons. And the applicability of some kinds of these factors to pharmaceutical formulations has also been studied.
Galectin is a generic name for an animal lectin specific for a lactosamine sugar chain, and is also called β galactoside-binding animal lectin or S-type lectin. Galectin is confirmed to exist in the cells of animal tissues ranging from lower Invertebrata such as nematode and sponge to bird and human. Some species of this type of lectin had been found, and in 1994, these were proposed to be generically called “galectin” (S. H. Barondes et al, Cell, 76, 597-598, 1994). Up until now, galectin-1 to galectin-11 have been reported as members of the galectin family. It has been reported that the actions of these galectins are associated with cell growth and cell adhesion, but these physiological functions are still unknown (J. Hirabayashi et al., J. Biochem., 119, 1-8, 1996; N. L. Perillo et al., J Mol Med., 76, 402-412, 1998). With regard to galectin-1, the structures of a large number of animal-derived forms have been determined (human-galectin-1: J. Hirabayashi et al., J. Biochem., 104, 1-4, 1988; J. Hirabayashi et al., Biochim. Biophys, Acta., 1008, 85-91, 1989, rat galectin-1: L. B. Clerch et al., Biochemistry, 27, 692-699, 1988, mouse galectin-1: T. J. G. Wilson et al., Biochem. J., 261, 847-852, 1989, bovine galectin-1: W. M. Abbot et al., Biochem. J., 259, 283-290, 1989). Information regarding the galectin-1 gene and protein, and a remedy for autoimmune diseases are disclosed in Japanese Patent Application Laying-Open (kokai) No. 2-84192 (Title: Mammalian 14-β-gal Lectins, Applicant: Ideon Corporation) and International Publication WO94/11497 (Title: Method of causing selective immunosuppression using HL-60-related lectins, Applicant: Incyte Pharmaceuticals Inc.) However, there is no description regarding galectin-1 as a remedy for neurodegenerative diseases such as nerve injury and neuropathy in the above documents.
On the other hand, as stated below, there are some reports on the relation between galectin-1 and the nervous system: galectin-1 expresses in a dorsal spinal nerve root ganglion cell at the development stage of a sensory nerve (J. Dodd, et al. J Exp Biol. 124, 225-238, 1986; M. A. Hynes, et al. J. Neurosci., 10, 1004-1013, 1990); galectin-1 is associated with the aggregation of nerve cells or the development of neurites, as a cell adhesive substrate, in a dorsal spinal root ganglion cell (R. L. Outenreath et al., J. Neurocytol., 21, 788-795, 1992); galectin-1 expresses in a rat olfactory nerve cell at the development stage, and is associated with the development of axons as a cell adhesive substrate (N. K. Mahanthappa, et al. Development. 120, 1373-1384, 1994; E. H. Raabe, et al. Brain Res Dev Brain Res. 101, 187-196, 1997); galectin-1 is associated with the development of axons in a culture system comprising mouse olfactory nerve cells, as a cell adhesive substrate (A. C. Puch, et al. Dev. Biol. 179, 274-287, 1996); there are reports on the distribution of galectin-1 in some kinds of nerve tissues (R. Joubert et al, Dev. Neurosci. II, 397413, 1989; S. Kuchler et al, Dev. Neurosci., II, 414-427, 1989), etc. These reports on galectin-1 are limited to discussion of its distribution in nerve tissues or its function as an adhesive substrate of nerve cells, and contain no description of galectin-1 as a factor that promotes nerve regeneration by acting on a nerve cell or a paraneural system, such as do neurotrophic factors or cytokines which act on the nervous system. Furthermore, it has been found that human galectin-1 has 6 cysteine residues in a molecule thereof, and that this protein has β-galactoside binding activity in a state where this protein is reduced, i.e., the cysteine is free, whereas this protein does not have that activity in a state where this protein is oxidized, i.e., a SS bond is formed. All of the above-mentioned laying-open applications or reports regarding the nervous system describe regarding the existence and function of galectin-1 as a lectin. However, there is no report at all in respect of the physiological action of galectin-1 on nervous systems as a nerve regeneration promoting factor or a nerve survival-support factor in a state where this protein is oxidized, i.e., SS bonds are formed and there is no lectin activity.
Not only the above stated biological properties, but also physicochemical properties are also greatly different between oxidized galectin-1 and reduced galectin-1. Since the formation of a SS bond by oxidation means that one cystine residue is formed from two cysteine residues, accordingly the oxidized protein loses two hydrogen atoms per SS bond, i.e., 2 Dalton (Da) of molecular weight are decreased. Human galectin-1 may form 3 couples of SS bond, and in such a case, the protein loses 6 Da of molecular weight. The difference of molecular weight can be determined by measuring the molecular weight with a high precision mass spectrometer. Furthermore, since the higher order structure of protein is greatly changed by the formation of SS bond, and both the steric dimensions of molecule and amino acid residues existing on a molecular surface are changed, the mobility on SDS electrophoresis and the elution time in reversed phase chromatography, ion exchange chromatography and the like are also changed. By examining physicochemical properties, it is possible to distinguish whether galectin-1 is in an oxidized state or in a reduced state.
In neuropathy, the degeneration and deciduation of nerve tissues, the transection and regression of axons, and the like occur. So, as an effective remedy for preventing or treating this disease, a neurotrophic factor or the like which acts to control the degeneration of nerve tissues or apoptosis and promote the regeneration of neurites is desired. The study on the applicability of a neurotrophic factor group including NGF, CNTF, BDNF etc. to remedies has been proceeding. These neurotrophic factor groups are ones having a function of promoting neurite regeneration and supporting the survival, and these were mainly found in nerve cells isolated from fetal or young animals at the development stage. So, to allow expression of the actions of neurotrophic factors, it is necessary to allow the factors to directly act on nerve cells. However, the reactivity to factors may be different between nerve cells of adult animals and those of fetal or young animals. Furthermore, as distinct from its state in a culture plate, each nerve cell does not exist separately in vivo, but generally nerve cells attach with each other or are enclosed by paraneural system cells such as Schwann cell, so that they exchange information interactively to maintain their functions. Where nerves are regenerated after nerve injury occurred, nerve cells perform cross talk with other cells surrounding them to repair their functions. As is clear from the above descriptions, there is the problem of how the factor groups can be made to act on nerve cells on which they directly act, in other words, there is the problem of administration method, therefore the development of remedies for neuropathy faces difficulty.
Under these circumstances, using the organ culture system of nerve tissues which maintains its functional structure in vivo, the present inventors have continued a thorough study to find out a novel protein factor indicating activity which promotes neurite regeneration from a transected nerve end of nerve tissues and supports its survival, or a novel use of the known factors as remedies for treating neuropathy or nerve injury.
To sum up, the main purpose of the present invention is to provide galectin-1 which is effective for treating neuropathy involving nerve injury, nerve degeneration and hypofunction at nerve grafting and derivatives thereof, and remedies for neuropathy containing them as active ingredients.